Host-microbiome interactions in apical periodontitis: The endodontic microbiome in relation to circulatory immunologic markers.

AIM: To explore microbial differences in the endodontic infection of teeth with primary or secondary apical periodontitis (AP), with or without symptomatology. Additionally, to investigate if these differences are depicted in immunologic markers in blood. METHODOLOGY: Twenty-nine teeth with primary or secondary AP were extracted and cryo-pulverized. Blood was drawn from the subjects at three different time-points before and three time-points after the extraction in a time period of four months. The V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq. The microbial profiles were ordinated using principal component analysis and tested for differences between groups with permutational multivariate analysis of variance using the Bray-Curtis distance. If significantly different, the microbial profiles were further analysed using the LDA effect size (LEfSe) biomarker discovery tool. A broad panel of inflammatory mediators in blood was examined longitudinally in all subjects during the six visits with mixed models. The Spearman correlation between these mediators and the zOTUs was calculated, and significant correlations (p < .05) were used as input for significant analysis of microarrays (SAM) using MeV. RESULTS: After subsampling, the 467 zOTUs were classified into 9 phyla and 99 genera or higher level taxa. The predominant genus in the entire sample set was Fusobacterium with a relative abundance of 12.3%, followed by Prevotella (9.9%), Actinomyces (7.7%) and Streptococcus (6.7%). The microbiomes of the endodontic infections were significantly associated with endodontic status (primary/secondary infection; p = .015) as well as with the presence or absence of pain (p = .011). There was also a difference in the concentration of inflammatory mediators, namely, C-reactive protein, Interleukin (IL)-8, IL-10, IL-12p70, RANKL and TNF-α, depending on the existence of pain. In addition, the presence of specific bacteria (zOTUs) was correlated, positively or negatively, with the expression of several circulating inflammatory markers. CONCLUSIONS: The microbial profiles and the concentration-time relationship of systemic inflammatory mediators of primary endodontic infection differed from those of secondary, and of symptomatic from those of asymptomatic cases. The fingerprint of associations between the immunological and microbiological profiles differed between asymptomatic and symptomatic patients.

A critical analysis of research methods and experimental models to study intracanal medicaments.

In order to ensure predictable decontamination of the root canal system, chemo-mechanical preparation of the root canal space is sometimes supplemented with the use of intracanal medication. As microbial control of the root canal space is fundamental to the resolution of apical periodontitis, root canal disinfection strategies haven been researched intensively. The use of intracanal medication as a supplementary step to the chemo-mechanical preparation of the root canal space is one of them. Because of the costs and limitations of clinical research it is relevant and common practice to first evaluate alternative or new root canal disinfection modalities in laboratory studies. This involves the simulation of a root canal infection in a laboratory model, on which different disinfection strategies can be tested. When modelling the infected root canal, different levels of infection can be discriminated: suspended bacteria, microbial biofilms and infected dentine. This review describes the experimental models associated with these infection levels and critically appraises their value and methodological details. Suggestions for relevant research methods and experimental models are given, as well as some good practices for laboratory-based microbiological studies.

Single nucleotide polymorphisms as a predisposing factor for the development of apical periodontitis-An umbrella review.

BACKGROUND: The interaction between heredity and different environmental factors in the modification of apical periodontitis (AP) susceptibility and prediction of its progression remain poorly elucidated. OBJECTIVES: This umbrella review aimed to (i) analyse the available relevant systematic reviews in an attempt to determine the association between genotype and allelic distribution of different single-nucleotide polymorphisms (SNPs) and the development of AP, (ii) report deficiencies and gaps in knowledge in this area and (iii) present recommendations to conduct future clinical studies and systematic reviews. METHODS: A literature search was conducted using Clarivate Analytics' Web of Science, Scopus, PubMed and Cochrane Database of Systematic Reviews, from inception to October 2021, with no language restrictions, including a grey literature search. Systematic reviews with/without meta-analysis evaluating genotype and allelic distribution of different SNPs between adult patients with/ without AP were included. All other type of studies were excluded. The methodological quality was assessed using the A MeaSurement Tool to Assess systematic Reviews (AMSTAR)-2 tool. Two independent reviewers were involved in study selection, data extraction and appraising the included reviews; disagreements were resolved by a third reviewer. RESULTS: The current study includes five systematic reviews. Three reviews performed meta-analysis. Three reviews were graded by AMSTAR 2 as 'critically low' quality, whereas the other two were graded as 'low' and 'moderate' quality. Two reviews indicated that carriers of specific genotypes and alleles of tumour necrosis factor-alpha (TNF-α) -308 G > A and interleukin 1-beta (IL-1ß) + 3954 C/T gene polymorphisms are more susceptible to an acute and persistent form of AP. However, high heterogeneity was observed. DISCUSSION: The statistical heterogeneity within included systematic reviews was a consequence of clinical and methodological diversity amongst primary studies. Although some of the included reviews suggested that carriers of specific genotype and/or allele of TNF-α -308 G > A and IL-1ß + 3954 C/T SNPs are more susceptible to AP, their conclusions should be interpreted with caution. CONCLUSIONS: No candidate genes could be identified as a definitive genetic risk or protective factor for the development and progression of AP, and further high-quality genome-wide association studies are warranted.

Effects of three γ-alkylidene-γ-lactams on the formation of multispecies biofilms.

This study evaluated the inhibitory effects of lactams on Streptococcus mutans, Enterococcus faecalis, and Candida glabrata multispecies biofilm formation. γ-Alkylidene-γ-lactams 1, 2, and 3 [solubilized in 3.5% dimethyl sulfoxide (DMSO)] were tested. Glass coverslips were conditioned with either the lactams or 3.5% DMSO (control) for 1 h, inoculated with microbial cultures, and incubated for 48 h. To assess the effect of the lactams on biofilm formation, the following parameters were determined: the biofilm biomass (by both crystal violet staining and protein determination); the amount of insoluble polysaccharides of the extracellular matrix; and the number of viable and total cells [by both colony-forming unit counting and quantitative real-time PCR (qPCR)]. Data were analysed using one-way anova and post-hoc Tukey tests. Lactams 1, 2, and 3 promoted a statistically significant reduction in the amount of biofilm biomass, but only lactam 3 resulted in a statistically significant reduction in the number of attached viable E. faecalis. Both total protein content and the amount of extracellular polysaccharides decreased significantly. The effects of γ-alkylidene-γ-lactams 1, 2, and 3 on the inhibition of multispecies biofilm formation were evident by their ability to reduce the amount of protein and extracellular polysaccharides.

The effect of different sweeteners on the oral microbiome: a randomized clinical exploratory pilot study.

Introduction: The aim of the study was to evaluate the modulating effects of five commonly used sweetener (glucose, inulin, isomaltulose, tagatose, trehalose) containing mouth rinses on the oral microbiome. Methods: A single-centre, double-blind, parallel randomized clinical trial was performed with healthy, 18-55-year-old volunteers (N = 65), who rinsed thrice-daily for two weeks with a 10% solution of one of the allocated sweeteners. Microbiota composition of supragingival dental plaque and the tongue dorsum coating was analysed by 16S RNA gene amplicon sequencing of the V4 hypervariable region (Illumina MiSeq). As secondary outcomes, dental plaque red fluorescence and salivary pH were measured. Results: Dental plaque microbiota changed significantly for two groups: inulin (F = 2.0239, p = 0.0006 PERMANOVA, Aitchison distance) and isomaltulose (F = 0.67, p = 0.0305). For the tongue microbiota, significant changes were observed for isomaltulose (F = 0.8382, p = 0.0452) and trehalose (F = 1.0119, p = 0.0098). In plaque, 13 species changed significantly for the inulin group, while for tongue coating, three species changed for the trehalose group (ALDEx2, p < 0.1). No significant changes were observed for the secondary outcomes. Conclusion: The effects on the oral microbiota were sweetener dependant with the most pronounced effect on plaque microbiota. Inulin exhibited the strongest microbial modulating potential of the sweeteners tested. Further full-scale clinical studies are required.

Sodium chloride and potassium sorbate: a synergistic combination against Enterococcus faecalis biofilms: an in vitro study.

Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48-h E. faecalis biofilms were evaluated in colony-forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)(2) ] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double-hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections.

The Evaluation of the Effects of Two Probiotic Strains on the Oral Ecosystem: A Randomized Clinical Trial.

Introduction: In the current study, we evaluated the effectiveness of two well-defined probiotic strains, Lactobacillus paracasei LPc-G110 (CCTCC M 2013691) and Lactobacillus plantarum GOS42 (DSM 32131), during an experimental gingivitis challenge. The primary objective was to evaluate clinically the effectiveness of lozenges containing one of the two oral probiotic strains, compared with placebo lozenges, on the gingival bleeding (bleeding on marginal probing; BOMP change) after a two-week experimental gingivitis period. The secondary objectives were to assess the effects of the test products on gingival health (Modified Gingival Index; MGI), dental plaque accumulation and fluorescence, and the dynamics of immunological and microbiological aspects after the wash-in phase, followed by a two-week period refraining from oral hygiene and a two-week wash-out phase. Methods: This single-center challenge intervention study was a triple-blind randomized placebo-controlled clinical trial with three parallel groups. The full study population consisted of 117 healthy 18-55 years old human volunteers. Subjects were instructed to use one lozenge, 3 times daily after each meal, containing either L. plantarum, L. paracasei, or lozenges without probiotics (placebo group). After a 2-week wash-in period, the subjects were requested to refrain from any form of oral hygiene for 2 weeks. Results: There were no differences in the primary outcome (BOMP change) among the groups. However, gingival health (MGI) in individuals from the groups exposed to the test products recovered better from experimental gingivitis than the individuals in the placebo group (p = 0.021, one-way ANOVA). The two test products inhibited pro-inflammatory cytokine IL-1ß production, measured in saliva, during the experimental gingivitis period. Both test strains significantly reduced bacterial DNA in tongue samples and L. paracasei strain showed stronger microbiome-modulating potential than the L. plantarum strain. Conclusions: The two tested lozenges with the L. paracasei or L. plantarum strains did show potential for beneficial effects for the oral health of the host during experimental gingivitis to the oral ecosystem.

Apical Periodontitis Is Associated with Elevated Concentrations of Inflammatory Mediators in Peripheral Blood: A Systematic Review and Meta-analysis.

INTRODUCTION: Apical periodontitis (AP), except for the local known consequences, may also be a systemic burden. Circulating inflammatory mediators that are released to sustain the AP lesion can in theory harm other bodily tissues. The aim of this systematic review was to summarize the existing evidence on the influence of AP on the peripheral blood levels of inflammatory mediators and markers of systemic stress. METHODS: A search of MEDLINE-PubMed, Embase, and Cochrane was conducted up to and including February 2019 to identify studies in 5 different languages. The Newcastle-Ottawa Scale was used for quality assessment of the included studies. RESULTS: Twelve of the 20 included studies were case-control studies, and 8 were intervention studies. The data of all the included studies were analyzed descriptively, whereas the data of 11 studies were available for meta-analyses. The study designs were heterogeneous. Nevertheless, the meta-analyses revealed statistically significant differences in C-reactive protein, interleukin 6, and asymmetric dimethylarginine levels between AP subjects and controls in peripheral blood. In addition, the concentration of C3 complement fragment in peripheral blood was significantly lower after the treatment and resolution of AP than before. CONCLUSIONS: The existing literature indicates that AP adds on to systemic inflammation by elevating C-reactive protein, interleukin 6, asymmetric dimethylarginine, and C3 levels. In order to overcome the issue of large variation between study designs, future studies should have clear inclusion criteria, preferably larger cohorts, adequate follow-up of all subjects, and a thorough presentation of the data to enable further exploration of the possible burden of AP on general human health. Nevertheless, there is now stronger evidence that AP contributes to low-grade systemic inflammation.

Calcium Hydroxide Treatment Does Not Alter the Susceptibility of Enterococcus faecalis Biofilms to Sodium Hypochlorite.

OBJECTIVE: To investigate the influence of calcium hydroxide (Ca(OH)2) on susceptibility to disinfection with sodium hypochlorite (NaOCl) of biofilm bacteria. METHODS: Monospecies biofilms of eight Enterococcus faecalis strains were subjected to a 2-h challenge with Ca(OH)2. After a recovery phase, the biofilms were treated with a concentration of NaOCl that was lower than the minimum inhibitory concentration. In a metabolic assay, the efficacy of NaOCl disinfection in Ca(OH)2-challenged biofilms and unchallenged biofilms was evaluated. The data were analyzed with Mann-Whitney U and Kruskall- Wallis tests. A P value of less than 0.05 was considered statistically significant. RESULTS: There were marginal differences in susceptibility to NaOCl among the E. faecalis strains. After the Ca(OH)2 challenge, seven strains remained equally susceptible to NaOCl disinfection whereas one strain became more resistant to NaOCl (P=0.03). CONCLUSION: After a Ca(OH)2 challenge, in general E. faecalis remained equally susceptible to disinfection with NaOCl.

Effectiveness of EDTA and Modified Salt Solution to Detach and Kill Cells from Enterococcus faecalis Biofilm.

INTRODUCTION: Disruption of the matrix of endodontic biofilms will aid in their removal from a root canal. Therefore, the aim of this study was to investigate the efficacy of EDTA and a modified salt solution (MSS) to detach bacteria from biofilms. METHODS: Forty-eight-hour-old Enterococcus faecalis biofilms were grown on glass coverslips and then treated for 1 hour by immersion in 17% EDTA or MSS. Phosphate-buffered saline served as a negative control. Then, residual biofilm cells on the substrate and the detached cells in the supernatant were collected. Viability was verified by the colony-forming unit (CFU) counting method. Propidium monoazide (PMA) treatment in conjunction with quantitative polymerase chain reaction (qPCR) was also performed to detect the presence of E. faecalis 16S ribonucleic RNA genes. Data were analyzed using 1-way analysis of variance and Tukey or Kruskal-Wallis and Dunn tests. The Pearson R test evaluated the correlation between results from CFU and PMA (α = 5%). RESULTS: qPCR showed that EDTA detached 99% of biofilm cells, and MSS detached 94% of biofilm cells (both P < .001). In contrast to EDTA, MSS was highly antimicrobial. The treatment promoted an ample log 7 reduction of the attached cells (P < .001), and almost no live cells were detected in the supernatant (P < .001). Positive correlations between CFU and qPCR with PMA were observed (r = 0.959 and r = 0.729). CONCLUSIONS: EDTA detached cells in biofilms with a minor antimicrobial effect. Besides a great antimicrobial effect, MSS also detached biofilm cells. These dispersals of biofilms give insights into new endodontic biofilm removal strategies.

Additional disinfection with a modified salt solution in a root canal model.

OBJECTIVES: The aim of this study is to investigate the disinfecting properties of a modified salt solution (MSS) and calcium hydroxide (Ca(OH)2) in a non-direct-contact ex-vivo model. METHODS: Seventy-four single-canal roots infected with Enterococcus faecalis were treated with 1% sodium hypochlorite (NaOCl) irrigation or with NaOCl irrigation with subsequent dressing with MSS or Ca(OH)2. After removal of the dressings, the roots were filled with bacterial growth medium and incubated for seven days to enable the surviving bacteria to repopulate the root canal lumen. Growth was determined by sampling the root canals with paper points before treatment (S1), after treatment (S2) and incubation after treatment (S3). The colony forming units were counted at S1 and S2. At S3, growth was determined as no/yes regrowth. The Kruskal-Wallis, McNemar and χ(2) test were used for statistical analyses. RESULTS: At S2, in the NaOCl group, growth was found in 5 of 19 root canals. After the removal of MSS or Ca(OH)2 bacteria were retrieved from one root canal in both groups. At S3, repopulation of the root canals had occurred in 14 of 19 roots after sole NaOCl irrigation, 6 of 20 roots after MSS-dressing and in 14 of 20 roots after Ca(OH)2-dressing. MSS was more effective in preventing regrowth than Ca(OH)2 (P=0.009). CONCLUSIONS: The modified salt solution prevented regrowth in roots which indicates that it can eliminate persistent bacteria. Dressing the root canals with Ca(OH)2 did not provide additional disinfection after NaOCl irrigation.

An evaluation of the accuracy of labeling of percent sodium hypochlorite on various commercial and professional sources: is sodium hypochlorite from these sources equally suitable for endodontic irrigation?

INTRODUCTION: The shelf life of sodium hypochlorite (NaOCl) is limited, and a previous article showed that there can be a discrepancy between the expected concentration of free available chlorine (FAC) and the actual FAC concentration in NaOCl solutions intended for endodontic irrigation. The current study investigates the FAC content of domestic and professional NaOCls and evaluates the influences of dilution and storage on FAC concentration. METHODS: First, domestic and professional NaOCls not obtained from manufacturers were iodometrically titrated. Then, NaOCls were diluted with demineralized water or tap water and stored at 4°C or 18°C and analyzed at baseline and 2 and 22 weeks. Statistical analyses included paired samples, independent samples t tests and repeated multivariate analysis of variance. Correlations were calculated with the Pearson or Spearman rank correlation test. A P < .05 was considered significant. RESULTS: Label specifications of domestic NaOCl were very imprecise (ie, <5% NaOCl). Domestic NaOCl contained 1.8%-3.5% NaOCl (w/v). Professional NaOCl varied from 14.3% relative less FAC than specified on the label to 23.5% relative more FAC than specified. After 22 weeks, the relative average loss of FAC in all conditions was 5.4% FAC (P = .002). Dilution, diluents, or storage temperature had no effect on the decline of FAC caused by aging. CONCLUSIONS: There is a great variation in NaOCl concentrations, with domestic NaOCl being the least accurate. NaOCl can be stored up to 5 months. The FAC concentration of domestic NaOCl is unpredictable, and, therefore, it appears less suitable for clinical application as root canal irrigant.

Potential of calcium to scaffold an endodontic biofilm, thus protecting the micro-organisms from disinfection.

Biofilms in the root canal of a tooth (endodontic biofilm) can induce and sustain apical periodontitis which is an oral inflammatory disease. Still, little is known about the composition of the endodontic biofilm. Studies on biofilms in root canals focus on the identification of the microbial species, but the majority of the biofilm consists of matrix material. Environmental aspects determine the structure of the biofilm and extracellular matrix. Calcium is involved in biofilm formation and activity at three levels. Firstly in cell-environment; calcium may 'condition' the surfaces of support and bacterial cells. Secondly, in cell-cell interaction; calcium plays a role in build up of biofilm structures. Typically, calcium ions act as 'cation bridges' between polysaccharides originating from different cells. Thirdly, within cells, calcium is required for certain biochemical reactions in bacteria and some bacterial physiological activities. Because calcium is present in the root canal, it could play a significant role in the organization of the biofilm. Chelators, already used in endodontics to remove the smear layer by disintegration of the structural cohesion calcium bonds, could weaken the biofilm matrix by removing calcium from the extracellular matrix thus disturbing its coherence. Subsequently, this disruption could increase the efficacy of disinfecting agents.